In the last decade many details have been clarified which contribute to understanding the role of oxygen free radicals and other oxygen metabolites in biology and medicine. Reactive oxygen species have been incriminated as deleterious species which are responsible for the biological damage induced by oxidative stress in many clinical cases, and today these metabolites are considered to be responsible for the oxygen toxicity in mammals, bacteria and plants. Humans are exposed to many types of oxygen metabolites from both endogenous and exogenous sources. During the reduction in stages of the oxygen molecule in the mitochondria for energy generation, active oxygen species are produced. These metabolites, which include the superoxide radical (O.sub.2.sup.-.cndot.), hydrogen peroxide (H.sub.2 O.sub.2) and the hydroxyl radical (OH.sup..cndot.), can leak to the immediate surroundings and may cause biological damage. Other internal sources for reactive oxygen species are enzymes which produce these metabolites as a result of their catalytic activity. The production of oxygen reactive metabolites can also occur in many other systems. Phagocytes, for example, are known for their ability to produce the superoxide radical and other reactive species. Other species also participate in the defense mechanism against invaders. This process, although necessary, can lead to biological damage in the surrounding area.
Exposure of humans to free radicals is not limited to the endogenous oxygen free radicals, but also includes exogenous sources. Various chemicals in agricultural use can serve as free radical generation systems, as in the case of the herbicide Paraquat. Other chemicals (aloxan, isouramil, cigarette smoke, air pollutants, carcinogenic and mutagenic compounds and many drugs) can generate oxygen free metabolites and cause biological damage. Free radicals have been shown to play an important role in the initiation and pathogenesis of, inter alia, inflammations, autoimmune diseases, brain degenerative diseases (Parkinson, Wilson, epilepsy), and eye diseases (cataract and retinopathy). These reactive species have also been demonstrated to be involved in ischemic and post-ischemic damage to the heart, brain and gastrointestinal tract. Recently it has been suggested that oxygen free radicals take part in cancer, aging and aging-related diseases.
Antioxidants
Exposure of the cells to continuous efflux of oxygen free radicals and reactive species led to the adaptation of the cells to live in an aerobic atmosphere. This adaptation process included the development of several lines of defense (antioxidants) against the damage induced by these metabolites. The broad definition of an antioxidant includes compounds which can cope with oxidative stress in various mechanisms. These mechanisms include: compounds which donate hydrogen to the damaged target, compounds which can scavenge free radicals, compounds which can bind the oxidants and remove them from the target, compounds which can convert reactive species to nonreactive metabolites, and reducing compounds which can react with oxidants. The various antioxidants may be classified into two main groups: the enzymatic group of antioxidants and the low molecular weight antioxidants (LMWA). The antioxidant enzymes include the enzymes superoxide dismutase, catalase and peroxidase. The LMWA include compounds which are not synthesized by humans but are present in the diet, such as ascorbic acid (vitamin C), tocopherol (vitamin E) and compounds which can be synthesized by humans such as glutathione (GSH), carnosine (an antioxidant present in the brain and muscle), uric acid and others. Most of the compounds in this group are reducing agents which can react with the oxidants.
Lipid Peroxidation Process
Among the various mechanisms that explain the oxidative damage of various biological systems, the lipid peroxidation process is of major importance. The occurrence of lipid peroxidation in biological membranes causes impairment of membrane functioning, decreased fluidity, inactivation of membrane-bound receptors and enzymes, and increased non-specific permeability to ions such as Ca(II). Peroxidation is initiated by the attack of any chemical species that has sufficient reactivity to abstract a hydrogen atom from a methylene carbon in the side chain. The hydrogen atom is a free radical and its removal leaves behind an unpaired electron on the carbon atom to which it was originally attached. The resulting carbon centered radical (L.sup..cndot.) can have several fates, but the most likely one in aerobic cells is that it will undergo molecular rearrangement, followed by a reaction with molecular oxygen to yield a peroxy radical (ROO.sup..cndot.). Peroxy radicals can combine with each other or they can attack membrane proteins, but they are also capable of abstracting hydrogen from adjacent fatty acid side chains in a membrane and in so doing propagate the chain reaction of lipid peroxidation. The result of this hydrogen atom abstraction is the production of lipid hydroperoxide (ROOH).
Evaluation of Lipid Peroxidation
Evaluation of the lipid peroxidation status in food products is important for the estimation of shelf life (for food products) and in biological systems for assessing the clinical condition of the organism. It has been shown that an increase in the oxidation of lipids correlates with the aging process and with pathological events in the cell. The measurement of the lipid peroxidation process and products in human materials is probably the evidence most frequently quoted in support of the involvment of free radicals reactions in tissue damage by disease. Oxidation of lipids can be measured at different stages, including (1) measurement of losses of unsaturated fatty acid; (2) measurement of primary peroxidation products; and (3) measurement of secondary carbonyls and hydrocarbon gases. Search of the scientific literature reveals that there is no method to evaluate lipid peroxidation of animal skin by a non-invasive procedure.
Evaluation of Antioxidant Activity
One of the major problems in this field of free radicals and antioxidants is the evaluation of the ability of a certain biological tissue or fluid (blood, CSF, saliva, sperm, etc.) to cope with the oxidative stress and to prevent the biological damage. The existing methods are based on the measurement and quantification of a specific compound or several compounds in body tissue or the determination of the concentration of a compound or several compounds following exposure of the tissue or the animal to oxidative stress. These methods are insufficient as they give only partial information on the tissue antioxidant status.
Antioxidant Activity of the Skin
The epidermis provides the first line of defense against oxidative stress and reactive oxygen species. It has been shown that the surface of the skin is equipped with a defending system which copes with the oxidative stress. The sources of the oxidative stress may be internal (such as infiltration of neutrophils and macrophages into inflamed skin or ischemic process) or external (inonizing radiation; UV light; photochemical reaction products such as superoxide radicals, hydrogen peroxide, hydroxyl radicals; products of the lipid peroxidation process or physical burns). The variety of the antioxidant compounds in intact skin have never been studied in detailed. Non-invasive methods for evaluating the LMWA were not available.